As the field of microfluidics continues to progress, microfluidic devices have become convenient platforms for processing and manipulating micro-objects such as biological cells. Some embodiments of the present invention are directed to improvements in microfluidic devices and methods of operating microfluidic devices in which the spatial orientation of a microfluidic device is manipulated to select, collect, isolate, and/or retain desired micro-objects within a microfluidic device. Some embodiments of the present invention utilize gravitational force, alone or in combination with dielectrophoretic (DEP) or optoelectronic tweezer (OET) forces, to accomplish such selection, collection, isolation, and/or retention.